Free radicals in biological milieu generally provoke destructive reactions, owing to their high reactivity and non-specific chemistry. Recently, however, two new classes of enzymes have been identified in which reactive radical species are generated during the normal course of catalysis. In the first of these classes, the radical site is an amino acid side chain; in the second, semiquinones derived either from elaborated amino acid side chains or from exogenous cofactors provide the radical center. These paramagnetic species appear to be integral to the proper function of these enzymes. Understanding the means by which these radicals are generated, stabilized, and directed to effective catalysis is a major, long-term objective of the project. The manner in which the unpaired electron-spin density is distributed on the radical center is one means by which radical reactivity appears to be controlled. The local protein environment around the redox active side chain is a second important factor in channeling the reactivity of theses species, and the extent to which they are shielded from solvent by the overall protein structure is a third. The controlling features-will be studied in the upcoming grant period in a variety of radical-generating and utilizing enzymes. The specific systems upon which we intend to focus include the following: Photosystems I and II in oxygen-evolving organisms; ribonucleotide reductase, prostaglandin H synthase, photolyase, methylamine dehydrogenase, methanol dehydrogenase and cytochrome bc1. The radicals generated by these enzymes include tyrosine, tryptophan, cysteine, and the semiquinones derived from tryptophan tryptophylquinone, pyrroloquinoline quinone, and ubiquinone. Work on some of these systems will be done exclusively at Michigan State; others will be studied collaboratively. The methods we will apply include techniques from microbiology, molecular biology, biochemistry, spectroscopy, and computational chemistry. EPR, ENDOR, and ESE spectroscopies provide the primary analytical tools and are intended to assess the unpaired electron spin-density distribution, the radical structure, the local environment around the radical site, and solvent accessibility. Tyrosine radicals are the best characterized thus far and will continue to be the primary focus of the proposed work. Thus far, radical-containing enzymes have been uncovered somewhat accidentally. By characterizing these species in detail in those enzymes in which they occur, we anticipate that general principles of their catalytic activity will emerge and lead to their identification in other enzymatic reactions.